Protein post-translational modifications are central to human complexity, and changes in post-translational modification of proteins are tightly associated with human disease. In this proposal, we will develop general approaches for the design of proteins responsive to post-translational modifications, including serine/threonine and tyrosine phosphorylation; tyrosine sulfation; and specific oxidation via tyrosine nitration, glutathione oxidation, and protein S-nitrosylation. Misregulation of protein phosphorylation plays an important role in numerous human diseases, including cancer, heart disease, Alzheimer's disease, and diabetes. New approaches to interrogate the activites of kinases and phosphatases will have potentially diverse applications in human disease. We are developing approaches employing design and synthesis to understand the phosphoproteome in order to allow the determination of kinase and phosphatase activities in diverse contexts and in a way that is potentially both spatially and temporally addressable. These approaches will be useful for the analysis of the activities of specific kinases and will be readily adaptable to provide a general approach to the detection of protein kinase activity and toward the activity of diverse protein postranslational modifications. Protein sulfation of extracellular tyrosines provides a critical mode for changes in cellular surface presentation and molecular recognition. Tyrosine nitration and protein S-nitrosylation are common post-translational modifications associated with oxidative stress. Changes in oxidative conditions in cells lead to changes in protein and cellular function. Specific aim 1: Development of protein motifs responsive to Ser/Thr and Tyr phosphorylation. We will develop novel, small, designed protein sequences (15-20 amino acids) whose structure is dependent on phosphorylation and which display luminescence only when phosphorylated. The approach will be generalized for application to a diverse series of kinases. Multiple designed motifs will be prepared which contain alternative kinase recognition sites, will utilize natural amino acids to enable their use as genetically encodable sensors of protein kinase activity, and will be incorporated into proteins and the luminescence tested in cellular models. Specific aim 2: Design of protein motifs responsive to Tyr sulfation. We will develop a general platform for the analysis of protein tyrosine sulfation. Specific aim 3: Design of proteins responsive to specific oxidation. We will develop encodable protein domains which are responsive to specific oxidative cellular conditions, including tyrosine nitration, glutathione oxidation, and protein S-nitrosylation. In addition, these approaches will allow the analysis of the changes in post-translational modification which occur in response to drugs, other small molecules, and other cues that change kinase, phosphatase, sulfotransferase, and oxidative activity.